Methotrexate (MTX) forms the cornerstone treatment of various autoimmune disorders including rheumatoid arthritis (RA), a debilitating disease affecting approximately 1% of the population. The cessation of purine, pyrimidine and protein synthesis following MTX is believed to produce anti-inflammatory effects through an activation of this prodrug into methotrexate polyglutamates (Dervieux et al., 2004). More precisely, these polyglutamated metabolites are retained intracellularly and inhibit de novo purine biosynthesis (i.e. AICAR transformylase) to release adenosine, a potent anti-inflammatory agent (Cronstein, 2005). Data indicate that the selective addition of glutamic residues onto MTX significantly potentiate the efficacy of this prodrug in vitro (Allegra et al., 1985) with the pentaglutamylated form of MTX being 2500-hundred fold more potent than MTX itself against AICAR transformylase (Allegra et al., 1987). Moreover, the notion that MTX's effects are produced by long chain MTXPGs versus short chains MTXPGs is supported by the observation that the number of glutamic residues on MTX is associated with the clinical effects of this prodrug in rheumatoid arthritis (Dervieux T et al., 2009b). Several clinical applications have been derived from the observations that MTXPGs are associated with the anti-inflammatory effects of MTX (Angelis-Stoforidis et al., 1999; Dervieux et al., 2004; Dervieux et al., 2005; Hornung et al., 2008), and the drug monitoring of low dose MTX therapy using the surrogate red blood cells can be helpful to assess exposure, compliance, and thus establish whether an appropriate dose of MTX is being administered to any given patients. (Dervieux T et al., 2009a). In particular, it is well recognized that approximately 5-10% of individuals do not metabolize MTX effectively, with virtually the totality of the drug excreted unchanged in the urine within the first few hours following the once weekly administration of the drug (Chladek et al., 1998).
However, under its current format there are several drawbacks with the usage of RBC MTXPGs in clinical practice. First, steady state concentrations of long chain MTXPGs in erythrocytes are only achieved after several months of therapy (Dalrymple et al., 2008) and while useful to address potential pharmacokinetics issues (e.g., rapid excretion phenotype described above), MTXPGs are not ideal pharmacodynamic markers indicating whether control of disease activity is achieved in any given patients. RBC MTXPGs have also poor predictive value before 3-4 months therapy which limit their utility, particularly given that significant clinical benefit from MTX in RA is usually observed in the first 6 weeks of therapy, at a time where very polyglutamation is detectable in erythrocytes (Kremer and Lee, 1986).
It follows that novel markers of MTX effects and exposure complementing MTXPGs measurements in the surrogate erythrocyte or target lymphocyte could be extremely useful to improve the performances of the drug monitoring assay of MTX therapy in patients suffering from cancer, inflammatory disease, and autoimmune disorders. Such method would be particularly useful early in the course of MTX treatment (1-4 weeks therapy) and provide a valuable tool to identify patients presenting defective MTX metabolism such as those with the rapid excretory phenotype (and hence no formation of MTXPGs). Currently, no method is available to directly or indirectly quantify MTXPGs early in course of MTX therapy (i.e, one week), or after a single dose of MTX, owing to the low concentrations of these metabolites in targets cells and the inherent sensitivity challenges associated with their detection.